X-Ray Crystallography and Biocrystallisation
The determination of the three dimensional atomic structure of matter is one of the most important areas of pure and applied research. This field, known as X-ray crystallography, utilizes the diffraction of X-rays from crystals in order to determine the precise arrangement of atoms within the crystal. The result may reveal the atomic structure of substances of different origin such as e.g. metal alloys, salt crystals as well as the structure of proteins and nucleic acid, e.g. deoxy- and ribonucleic acid (DNA and RNA, respectively). Crystallographers have made some of the greatest discoveries in the history of science.
One important and rapidly growing field of crystallography is macromolecular crystallography involving biocrystallization, which is the crystallisation of proteins, nucleic acids and macromolecular assemblies described in Giegé et al. (Prog. Crystal Growth and Charact., 30:237, 1995), Crystalisation of Nucleic Acids and Proteins (editors Ducruix, A., and Giege, R., Oxford University Press, 2000, ISBN 19-963679-6) and in Current approaches to macromolecular crystallisation (McPherson, A., Eur. J. Biochem. 189:1–23, 1990). The limiting step in this area of research involves the screening of optimal crystal growth conditions of a particular sample. Like all crystallisation, it is a multiparameter process involving the three classical steps of nucleation, growth and cessation of growth of the crystal. Though, crystallisation of macromolecules differ in a sense compared to growth of smaller molecules due to the much larger number of parameters involved and peculiar physical-chemical properties (table 1). As an example, their optimal stability in an aqueous medium might be restricted to a rather narrow temperature, if the protein is not thermostable, and pH range. But, their main difference from inorganic and organic molecules is the conformational flexibility and chemical versatility of macromolecules and, consequently, their greater sensitivity to external conditions. For a rational strategy in crystallisation, however, the conditions for growth have to be set in a rational way.
Table 1. Parameters Affecting the Nucleation, Crystallisation and/or Solubility of Macromolecules
Intrinsic Physico-chemical Parameters
    Supersaturation    Fluctuations of temperature and pH    Time and rate of equilibration and growth    Ionic strength and purity of chemicals    Diffusion and convection    Volume and geometry of samples and set-ups    Solid particles wall and interface effects    Density and viscosity effects    Pressure, electric, and magnetic fields    Vibrations and soundBiochemical and Biophysical Parameters    Sensitivity of conformations to physical parameters    Binding of ligands and specific additives    Properties of macromolecules and ageing of samplesBiological Parameters    Biological sources    Physiological state of organisms or cells    Bacterial contaminants    Purity of Macromolecules    Macromolecular contaminants    Sequence and conformational heterogeneities    Batch effects
Proteins are polymers of amino acids and contain thousands of atoms in each molecule. Considering that there are 20 essential amino acids in nature, one can see that there exists virtually an inexhaustible number of combinations of amino acids to form protein molecules. Inherent in the amino acid sequence or primary structure is the information necessary to predict its three dimensional structure. Unfortunately, science has not yet progressed to the level where this information can be obtained directly from the primary structure. Considerable advances are being made in the area of high field nuclear magnetic resonance. Still, the only method capable of producing a highly accurate three-dimensional structure of a protein is by the application of X-ray crystallography. This requires the growth of reasonably ordered protein crystals (crystals which diffract X-rays to at least 2.0 angstroms resolution or better).
To grow crystals of any dissolved compound the solution containing the molecule(s) have to be brought to a supersaturated state, which is thermodynamically unstable. The solute may develop into a crystalline or amorphous state when it returns to equilibrium. Supersaturation can be achieved by evaporation of the solvent or by abrupt changes in physico-chemical parameters like temperature, pH or pressure. Supersaturation is a prerequisite for formation of crystalline nuclei from which crystals grow. The solubility of a polyelectrolyte (e.g. a protein) is a complex function and it is not trivial to theoretically simulate and thereby predict the conditions for nucleus formation. Therefore, the solubility of a compound is experimentally determined from a solution in equilibrium with crystals.
Because of the complexity of macromolecules, particularly membrane spanning proteins, obtaining suitable crystals can be quite difficult. Macromolecules are extracted from complex biological sources and purification plays an extremely important role in crystallogenesis. Purity must be of “crystallographic grade” meaning that the macromolecule must be correctly folded as well as free of contaminants. Denatured molecules or macromolecules with micro-heterogeneities, such as chemical modifications, negatively affect crystal growth. Typically several hundred to several thousand experiments must be performed to determine crystallisation conditions. Large matrixes exploring pH, buffer type, precipitant type, protein concentration, temperature, etc are examined. This process is extremely time consuming and labour intensive.
The three dimensional structure of a protein determines its function and activity. Knowledge of the structure is therefore imperative in medical and pharmaceutical applications. New and increasing knowledge about the structure of a protein will lead to an increase ability to produce new and relevant drugs. In structural biology, the technical prerequisites to solve complicated structures have dramatically advanced, which makes structural analysis a much faster process than before. In one sector of this multidisciplinary field of research, however, the work is slowed. That is the crystallisation of proteins, protein-ligand complexes or complexes with nucleic acid whose structure or function is to be studied. In pharmaceutical industry, as well as academia, the aim is parallel determination of the genomic sequences and structures of the gene products, but in order to do this the production of crystals must be much faster. Due to the complex and multidimensional character of the crystallisation process, crystallisation of macromolecules has remained a time consuming handcraft that often requires vast amounts of precious and expensive materials.
The resulting three-dimensional structure produced from the protein crystals can have enormous implications in the fundamental understanding of molecular biology such as how enzymes perform various catalytic activities, switch on chemical pathways, or transport molecules within the circulatory system. In the past few years the determination of protein structures important as therapeutic targets has made a rational design possible for new and more efficient pharmaceuticals.
Recent advances in structural biology like syncroton beam sources, fast X-ray detectors, cryotechniques, and high-speed computer graphics has revolutionised the pace at which the three-dimensional structures can be determined. Still, however, the bottleneck has been the determination of conditions necessary to grow high quality protein crystals suitable for X-ray diffraction methods. In order for protein crystals to be suitable for structural analysis via X-ray diffraction methods, crystals in the order of about 0.1–0.2 mm in diameter or greater must be obtained.
Conventional Methods for Crystallization
At present, biologically active macromolecules, such as proteins, are crystallised by a variety of experimental methods. Among these many methods, there are three that are most commonly used in the art (Current approaches to macromolecular crystallisation, McPherson, A., Eur. J. Biochem. 189:1–23, 1990; Crystallisation of nucleic acids and proteins, Edited by A. Ducruix and R. Grieg, The Practical Approach Series, Oxford University Press, 1992; Protein and Nucleic Acids Crystallisation Methods—A companion to Methods in Enzymology, Academic Press, 1:1, August 1990).
In most laboratories, the vapour diffusion method is used. This is a method wherein a drop of a solution containing protein is applied to a glass cover slip or sitting bridge and placed upside down in a sealed chamber, such as a vapor diffusion chamber, where conditions lead to supersaturation in the protein drop and the initiation of precipitation of the protein crystal.
In another method, referred to as the dialysis method, the protein solution is contained within a semipermeable size exclusion membrane and then placed in a solution of appropriate pH, precipitant concentration, etc., as in the reservoir solutions prepared for the vapour diffusion technique. As the precipitant diffuses through the membrane into the protein compartment, the solubility of the protein is reduced and crystals may form. Both vapour diffusion and dialysis methods require extensive screening of numerous variables to achieve the desired results and is, thus, time consuming.
Still another method of protein crystal growth involves what is referred to as gel crystal growth. This method involves the placement of a gel into the end of small diameter glass capillaries. After the solutions have gelled, a protein solution is placed into one end (top) of the capillary and the other end is submerged in a solution of precipitating agent. If the conditions are appropriately selected, crystal growth occurs at a point in the gel where the protein and precipitating agent reach the proper concentrations as the solutions slowly mix by diffusion. Since this is a diffusion-limited process, it thus only occurs after an extended period of time, i.e. precipitation after several hours depending on the size and complexity of the molecule. For larger molecules, such as proteins or complex macromolecular assemblies this approach may be less suitable due to diffusional restrictions. Crystals grown by this method are often larger and of high quality. The approach here used to screen for the proper crystallisation conditions involves the use of numerous bottles of precipitant solutions containing glass capillaries. The method is thus cumbersome and time-consuming. Also, it has the disadvantage of that once the crystals are formed in the gels it is extremely difficult to remove them without damage.
However, the methods described above are usually troublesome, time-consuming and inefficient because current means of employing this technique to achieve crystal growth are somewhat primitive, whether conducted manually or through robotic devices, and involve long series of adjustments of conditions until a suitable experimental regimen is found. In typical screening methods under this process, it is generally required that someone varies the conditions of pH, buffer type, temperature, protein concentration, precipitant type precipitant concentration, etc., for each set of experiments. Still, even after adjusting for the myriad of conditions, most often only minute samples of the protein can be studied at one time. As presently carried out using currently available devices crystal growth methods such as the hanging drop method are tedious, time-consuming, and hard to carry out successfully and efficiently with reproducibility.
Crystal growth in chambers or devices with inner-walls often contributes to the difficulties with producing single and clean crystals. Most often, the crystal then is growing on the wall of the crystal chamber, producing a poly-crystal hard to remove from the wall and subsequently remove a single crystal from without disrupting the structure.
Different means of improving crystallisation conditions including speeding up the initial screening of the specific conditions needed for growing a particular crystal, has been described. In U.S. Pat. No. 5,641,681, a device and method for detecting optimal protein crystallisation conditions in a 1×g or a micro-gravity environment has been described. The method utilises a diffusion-limited process with at least one pair of dialysis chambers housing the crystallisation solutions in which the crystals are grown. Since the diffusion herein is defined by the quantity of gelling substance, it will affect the period of time sufficient to achieve equilibration for the growth of the crystal. The device is useful for screening of crystal growth conditions, and reduces experimental time to about 3–6 months as well as a technical 10-fold reduction in experimental set-ups.
Gravitational phenomena, including convection, sedimentation, and interactions of materials with their containers all affect the crystal growth process. If they are not taken into consideration they can have adverse effects on the quantity and quality of crystals produced. As a practical matter, convection and sedimentation can be completely eliminated only under conditions of low gravity attained during orbital flight. There is, then an advantage of performing crystallisation in space, i.e. below 1 g or at microgravity conditions. As an alternative, conditions for growing crystals under 1 g or in a micro-gravity environment, e.g. space, have been described. One problem during crystal growth, is interaction of materials with containers affecting the crystal growth, as mentioned above. This will lead to crystal growth onto the walls of the container or chamber, wherein the crystallisation experiment takes place, causing quality problems and difficulties when removing them from the walls. U.S. Pat. No. 5,173,087 describes a process for controlling where the nucleation takes place in space by the use of a cooling chamber wherein the crystals are nucleated only at specific locations, i.e. not on the container walls and in controlled numbers only.
Another way of producing an environment below 1 g without performing experiments in space has been described. Different types of levitation systems, e.g. electrostatic or acoustic drop levitators are known and described in e.g. Rhim et al. (J. Chryst. Growth; 110:293, 1991). This allows a containerless method that is clean in two senses. First, the sample is not in physical contact with container or chamber walls that might induce uncontrollable nucleation. Secondly, through software programming of control forces, a sample drop can be isolated from much of the gravity forces as well as oscillatory and impulsive forces known to reside in laboratories, such as e.g. a space laboratory.
Nucleation
Crystallisation aims at establishing a phase separation where a solid phase, e.g. a protein crystal, shall be at equilibrium with a liquid phase. A protein dissolved in buffer must he brought to form a three-dimensional lattice. Practical experience shows that different types of precipitation agents, which change the dielectric constant of the solution, can bring this about. The crystallisation process has three major stages; nucleation, growth and cessation of growth. Crystallisation starts by a nucleation phase, i.e. the formation of the first ordered aggregates, which is followed by a growth phase. Cessation of growth can have several causes. Apart from the obvious ones, like depletion of the macromolecules from the crystallisation medium, it results from growth defects.
The most difficult phase to control in a crystallisation experiment is the nucleation process. The kinetics of nucleation is an exponential function of the degree of supersaturation and a critical value must reach before nuclei can build up. This is when the nucleation point is reached. A critical size of an aggregate is required for a crystal to grow, as seen in FIG. 1. It is an unstable state where physical condition is difficult to measure accurately but forms the basis for crystal nuclei to form.
The standard procedure when set up a crystallisation experiment with an unknown protein is to use commercially available buffer solution that empirically have been shown to work as crystallisation media for other proteins. The number of solutions can be up to 90–100 with different mixtures of precipitation agents at different pH and with additives like salts and polymers. Since a suitable level of protein concentration also needs to be established and different physical parameters, e.g. temperature, needs to be tested, this results in a very large amount of experiments that require huge amounts of sample, i.e. a macromolecule such as a protein. nucleic acid.
Limitations and Future Perspectives
The future perspectives of using small-scale analyses and rapid crystallisation of proteins are enormous in genomics, such as the HUGO (human genome organisation) project, where thousands of gene products will be cloned, expressed and characterised. Attempts to determine the three-dimensional structure of new proteins is part of such a thorough analysis, and protein crystallisation is a necessary step. The protein may further be introduced into single cells in order to study physiological effect(s) and function(s).
Joint efforts by international consortia are made world wide to determine the three dimensional (3D) structures of all proteins eventually identified. This involves research institutions as well as pharmaceutical industry. Protein crystallographers aim at determining high resolution structures in massive numbers at highly efficient synchrotron beam sources, for instance at Stanford (see http://www.jcsg.org/scripts/prod/home.html). This implies that new means and methods are developed meeting the demand for quick prediction of the physical-chemical conditions required for the formation of single crystals of individual proteins or macromolecular complexes used in X-ray experiments. Crystallisation screening kits for proteins as well as nucleic acids are commercially available (Hampton Research, Laguna Niguel, Calif., USA, see www.hamptonresearch.com). Also robots exist, which minimises human intervention in the practical work of setting up large-scale crystallisation trials. However, scientists lack means and methods, which rapidly screen for the crystal nucleation step, a prerequisite for the growth of crystals in the first place.
Problems that have limited the use of high resolution X-ray crystallographic methods in the determination of the three-dimensional structures of macromolecules, e.g. protein molecules is the time-consuming step of determining specific nucleation conditions of a protein. Given the nucleation conditions, a proper crystal may easily be made from the macromolecule. It is thus highly desirable in light of the recent advances in the field of protein crystallography to develop highly efficient, simple, and effective methodologies and means for obtaining the desired conditions for the growth of high quality protein crystals for X-ray crystallography, and yet which can also avoid the problems associated with the prior art methodologies and means, and in this respect, the present invention addresses this need and interest.